Heterogeneous nuclear ribonucleoprotein F/H proteins modulate the alternative splicing of the apoptotic mediator Bcl-x
Heterogeneous nuclear ribonucleoprotein F/H proteins modulate the alternative splicing of the apoptotic mediator Bcl-x
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DOI：
10.1074/jbc.m501070200
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发表时间：
2005-06-17
影响因子：
4.8
通讯作者：
Chabot, B
中科院分区：
文献类型：
--
作者：
Garneau, D;Revil, T;Chabot, B
x是Bcl-2蛋白家族的成员，是细胞凋亡的关键调节因子。Bcl-x前mRNA被选择性剪接以产生Bcl-x（S）和Bcl-x（L），这两种亚型分别与促进和预防细胞凋亡相关。我们已经调查了这两个剪接变异体的生产中涉及的一些元素和因素。使用人Bcl-x小基因的缺失诱变鉴定了外显子2中调节人HeLa细胞中Bcl-x 5 '-剪接位点选择的两个区域。一个区域（B3）位于Bcl-x（L）5 '-剪接位点的上游，并在细胞和剪接提取物中强制产生Bcl-x（L）。另一个区域（B2）位于Bcl-x（S）的5 '-剪接位点的下游，并且有利于体内和体外Bcl-x（S）的产生。富含30个核苷酸的G元件（B2 G）负责B2元件的活性。我们发现，重组异质核核糖核蛋白（hnRNP）F和H蛋白结合B2 G，和突变的G伸展废除结合。此外，向HeLa提取物中添加hnRNP F以依赖于B2 G中G延伸的完整性的方式改善了Bcl-x（S）变体的产生。与体外结果一致，靶向hnRNP F和H的小干扰RNA介导的RNA干扰降低了质粒衍生的和内源性产生的Bcl- x转录物的Bcl-x（S）/Bcl-x（L）比率。我们的结果证明了hnRNP F/H蛋白在促凋亡调节因子Bcl-x（S）的产生中的积极作用。
x is a member of the Bcl-2 family of proteins that are key regulators of apoptosis. The Bcl-x pre-mRNA is alternatively spliced to yield Bcl-x(S) and Bcl-x(L), two isoforms that have been associated, respectively, with the promotion and the prevention of apoptosis. We have investigated some of the elements and factors involved in the production of these two splice variants. Deletion mutagenesis using a human Bcl-x minigene identifies two regions in exon 2 that modulate Bcl-x 5'-splice site selection in human HeLa cells. One region (B3) is located upstream of the Bcl-x(L) 5'-splice site and enforces Bcl-x(L) production in cells and splicing extracts. The other region (B2) is located immediately downstream of the 5'-splice site of Bcl-x(S) and favors Bcl-x(S) production in vivo and in vitro. A 30-nucleotide G-rich element (B2G) is responsible for the activity of the B2 element. We show that recombinant heterogeneous nuclear ribonucleoprotein (hnRNP) F and H proteins bind to B2G, and mutating the G stretches abolishes binding. Moreover, the addition of hnRNP F to a HeLa extract improved the production of the Bcl-x(S) variant in a manner that was dependent on the integrity of the G stretches in B2G. Consistent with the in vitro results, small interfering RNA-mediated RNA interference targeting hnRNP F and H decreased the Bcl-x(S)/Bcl-x(L) ratio of plasmid-derived and endogenously produced Bcl- x transcripts. Our results document a positive role for the hnRNP F/H proteins in the production of the proapoptotic regulator Bcl-x(S).